DNA-based immunization may be of prophylactic and therapeutic value for hepatitis C virus (HCV) infection. In efforts to improve the immunogenicity of a plasmid expressing the second envelope protein (E2) of HCV, we evaluated in mice the role of the antigen localization and demonstrated that membrane-bound and secreted forms induced higher titers of E2-specific antibodies, as well as earlier and higher seroconversion rates, than the intracellular form, but all three forms induced strong CTL. We also investigated whether E2-specific antibody responses could be enhanced by CpG optimization of the plasmid backbone and showed that removal of neutralizing CpG dinucleotides did not have a significant effect but addition of 64 immunostimulatory CpG motifs significantly enhanced anti-E2 titers. These results may have implications for the design and development of HCV DNA vaccines.